Techniques for assessing dissolution of pharmaceutical formulations were originally introduced by regulatory authorities in the pharmaceutical industry in an attempt to characterize release profiles of formulations with low solubility in aqueous media. However, the growing demand for controlled use of pharmaceutical formulations has resulted in the adoption of dissolution testing for most formulations for quality control and regulatory purposes. Furthermore, dissolution testing is being used in the formulation development process in order to determine rate of release of the active compound from the formulation, and other parameters relating to formulation performance, to develop optimal dosage forms and to establish in vitro-in vivo correlations (IVIVC).
When assessing dissolution of a pharmaceutical formulation in an in vitro system, it is normally desirable to achieve high in vitro-in vivo correlation (IVIVC). An in vitro system producing data that closely correlates with dissolution and absorption data obtained in vivo would be beneficial in the pharmaceutical industry as a tool for various applications including dosage form development and scale-up, production scale-up, lot-to-lot bioequivalence testing, testing of new strengths, testing of minor formulation changes, testing after changes in the site of manufacture, and as a reference for bioequivalence requirements.
As dosage forms become more advanced, dissolution testing must become more rigorous to impart a fundamental understanding of how much of a pharmaceutically active compound is available at the absorption site(s) at specific times. Furthermore, establishing relationships between dosage form and availability of a pharmaceutically active compound at the absorption site(s) and systemic blood levels of a pharmaceutically active compound will allow investigators to optimize the in vivo performance of a pharmaceutically active compound through specialized delivery techniques.
Dissolution technology which allows determination of IVIVC for pharmaceutically active compounds which exhibit poor membrane permeability characteristics, which undergo extensive intestinal metabolism, or which require specialized transport systems for absorption has not yet been developed. Techniques for correlating in vitro and in vivo dissolution data have heretofore been limited to accounting for factors such as interactions with mucins, bile salts, digestive enzymes, food effects, ionic strength, and pH. Factors such as gastrointestinal transit of a dosage form may be accounted for by temporal displacement of the absorption and dissolution data. Discrepancies between in vitro and in vivo values of dissolution and absorption have previously been corrected for by transformation of data, such as by applying intestinal weighting functions, which transformations may not allow for physiological interpretation. There is currently a need for a system which combines dissolution technology with a biological intestinal absorption model, such as cell culture, for use in formulating and testing dosage forms of pharmaceutical formulations. Advantageously, such a system would more closely resemble the physiological system compared with existing dissolution methods.
Human intestinal epithelial cell culture has gained wide acceptance for delineating mechanisms of absorption of pharmaceutically active compounds. Conventional cell culture techniques allow for correlation of flux of a compound across an epithelial layer with passive transcellular or paracellular transport, carrier mediated transport, and active transport.
Actively absorbed compounds or those absorbed through carrier-mediated or energy requiring mechanisms usually have a saturable component to their absorption. Current cell culture techniques can delineate the details of absorption pathways as well as carrier affinity and capacity. Further, cell culture may be used to describe efflux systems, such as the p-glycoprotein mediated efflux system, which reduces the absorption of certain compounds. Intestinal metabolism of a compound may occur and will reduce the overall absorption of the compound. This information is currently used to explain discrepancies in IVIVC by transforming the kinetic models to include saturable Michaelis-Menton kinetics, or alternatively to include metabolic or efflux kinetics to improve IVIVC. Current cell culture techniques conducted in 2-dimensions across horizontal filters do not account for temporal displacement of a pharmaceutically active compound due to gastrointestinal transit and other in vivo conditions. Conventional cell culture techniques allow for interpretation of absorption by transformation of in vivo data, but do not yield adequate IVIVC, except in the assessment of passively absorbed compounds. Further, transport studies using cell culture describe the compound in solution, and thus do not account for dissolution or diffusion from a dosage form, or account for the effect of a formulation or dosage form on absorption of the compound of interest.
There is a need for the integrated assessment of in vitro dissolution of a pharmaceutical formulation and absorption of an active compound therefrom. These two parameters have conventionally been considered and assessed separately. In vivo, the appearance of a pharmaceutical compound in the blood stream results from inter alia the dissolution of the formulation and absorption of the compound. In the case of compounds which are passively absorbed, the relative rates of dissolution and absorption determine which is the rate-limiting factor in the appearance of a compound in the blood stream. With the advent of more complex dosage forms, especially for compounds and formulations which interact with epithelial surfaces for absorption (eg. bioadhesive systems), metabolism, or efflux (i.e. those not passively absorbed), more advanced systems are required to assess the formulation impact on dissolution and absorption. Advantageously, an integrated system of dissolution and absorption will provide improved IVIVC without the inconvenience of error associated with predictive models using transformed data.
Dissolution technology is known and described in, for example, U.S. Pat. No. 4,681,858 (Chaudhari et al.), which discloses a dissolution cell and method for determining the in vitro release of a pharmaceutically active compound from a dosage form, such as from a suppository. The dissolution cell allows for assessment of the rate of release of the compound of interest into the dissolution medium. Also, U.S. Pat. No. 5,589,649 (Brinker et al.) discloses a dissolution testing apparatus which incorporates a plurality of test vessels and uses heat and mechanical stirring to dissolve dosage forms in each test vessel.
Conventional cell culture methodologies incorporate flasks, plates or wells to grow cells or cell monolayers. These techniques incorporate agitation or turbulent mixing to ensure circulation of the medium to the growing cells. Other types of cell culture chambers have been developed. For example U.S. Pat. Nos. 5,026,650 and 5,153,133 (Schwartz et al.) disclose a bio-reactor consisting of a horizontally rotating cell culture system with a coaxial tubular oxygenator. Cell growth occurs on microcarrier beads suspended in media while the culture vessel rotates about a shaft, which provides continuous oxygenation through a membrane. U.S. Pat. No. 5,153,131 (Wolf et al.) discloses a horizontally rotating cell culture chamber with a dialysis membrane for exchanging cell waste products with fresh nutrients while cells are maintained in suspension. Employment of a horizontally rotating culture chamber, such as those described in U.S. Pat. Nos. 5,026,650, 5,153,133 and 5,153,131 provides good mixing of incubation media without agitation or turbulent mixing which can be damaging to cultured cells.
Conventional models used to assess absorption or permeability of a pharmaceutically active compound assume that no metabolism of the compound occurs within the gut wall (Chiou, Int. J. Clin. Pharm. Ther. 1994;32(9):474). However, the present invention could possibly be used to evaluate gut metabolism and the effect upon absorption of a compound. Additionally, conventional models assume that a compound, once absorbed, is immediately cleared from the basal side of the gut. This assumption does not account for conditions which may delay basal clearance. For example, anesthetics may decrease blood flow to the gut, thereby slowing basal drug clearance.
U.S. Pat. No. 5,518,915 (Naughton et al.) teaches a three dimensional mucosal cell and tissue culture system in which cells derived from a desired tissue are grown on a support matrix. The metabolism of, for example, a pharmaceutically active compound by the cells in this three-dimensional culture can be assessed. However, Naughton et al. does not incorporate dissolution of a pharmaceutical formulation prior to assessment of metabolism or absorption of a compound by the cultured cells.
U.S. Pat. No. 5,525,305 (Minekus et al.) discloses an in vitro model of the digestive tract. The system comprises tube-like chambers made of flexible material which are connected for the flow of gases or liquid therethrough. The contents of the chambers simulate gastric fluids and may include intestinal enzymes, acids, etc. This system is useful for in vitro assessment of digestion and exchange of low-molecular weight components through permeable membranes, but cannot assess biological parameters such as absorption across a membrane and does not incorporate cultured cells.
Canadian Patent Application No. 2,201,159 (Myers et al.) teaches an artificial liver apparatus and method which uses isolated hepatocytes which may be attached to inert carriers and suspended in cell culture medium to allow for purification of a biological fluid such as blood. As the biological fluid passes through the artificial liver apparatus and is exposed to hepatocytes, the hepatocytes can absorb and metabolize components of the biological fluid in a manner similar to the function of the liver in vivo. This system uses isolated cells as a model of in vivo hepatic metabolism but does not incorporate dissolution technology.
U.S. Pat. No. 4,667,504 (Hobson) discloses a flow-through device for determining the penetration rate of chemicals across a biological membrane in vitro. The apparatus comprises two chambers, one containing media including a test chemical, and the other containing media in which the test chemical appears when transported across the biological membrane which is suspended between the chambers. The media in each chamber may be sampled and analysed for concentration of the test chemical. However, this method does not incorporate dissolution methodology.